Compulsive Alcohol Drinking and Cortical Extracellular Matrix

酒精使用障碍（AUD）的几个诊断标准涉及饮酒，尽管有负面后果， 这意味着尽管失去了工作，破坏了关系，或者增加了伤害的风险， 自己。大脑中控制在危险情况下饮酒决定的一个区域是岛叶皮层。 过量饮酒引起的大脑神经元功能的变化可能会导致这种危险的转变， 或厌恶性饮酒。我们的初步数据表明， 被称为神经元周围网络（PNNs）的结构可能与抗厌恶饮酒有关。PNN在 皮质区域主要围绕快速尖峰GABA能中间神经元，其表达钙结合 小清蛋白（PV）。这些神经元对认知很重要，并严格控制着大脑的放电。 兴奋性皮质投射神经元。该提案的总体目标是：1）检查蜂窝和 长期酗酒后大鼠延髓PV神经元PNNs的分子变化 小鼠，以及2）确定操纵PV神经元和PV神经元上的PNN的行为后果 能抵抗厌恶的饮料在具体目标1中，我们将研究PNN的结构， 采用免疫细胞化学方法，检测酗酒后不同时间点， PNN基因和蛋白的表达，并测量蛋白酶（MMP和ADAMTS）的活性 金属蛋白酶家族）调节PNN的重塑。在具体目标2中，我们将衡量 在狂饮后，有和没有PNNs的PV神经元上的突触密度和体积 免疫细胞化学方法和超分辨率显微镜。此外，我们将测量兴奋性， 用电生理学方法观察了酗酒后这些神经元的突触变化。所有这些 两个具体目标将提供有关PNN发生的变化及其 相关的突触后，长期酗酒。在本提案的第三个具体目标中， 我们将通过敲低两种特异性PNN蛋白的表达来破坏PV神经元上的PNN， 基因Acan和Bcan使用病毒递送的短发夹RNA。病毒将被直接注入 在PV神经元中表达Cre重组酶的小鼠（PV-Cre）的定位细胞类型特异性基因的鉴定 击倒。这些小鼠将被测试单独的乙醇和乙醇的厌恶溶液的消耗 含有奎宁的水溶液，以确定PNN破坏对抗厌恶饮用的影响。最后我们将 通过病毒递送设计受体直接操纵岛叶PV神经元的活性 由设计师药物激活（DREADDs），以确定这些神经元在抵抗厌恶乙醇中的作用 消费这些研究将提供关于PV发生的细胞和分子变化的信息 大脑中的神经元驱动从狂饮到高风险饮酒的转变，并可能导致新的 治疗厌恶性饮酒的治疗策略。

Several criteria for diagnosis of alcohol use disorder (AUD) involve drinking despite negative consequences, meaning the decision to drink despite the loss of a job, damage to relationships, or increased risk of harm to oneself. One region of the brain that controls the decision to drink under conditions of risk is the insular cortex. Changes in neuronal function in the insula caused by excessive drinking may drive the transition to this risky, or aversion-resistant alcohol drinking. Our preliminary data indicates that specialized extracellular matrix structures, known as perineuronal nets (PNNs), may be involved in aversion-resistant drinking. PNNs in cortical regions primarily surround fast-spiking GABAergic interneurons that express the calcium-binding protein parvalbumin (PV). These neurons are important for cognition and tightly control the firing of excitatory cortical projection neurons. The overarching goals of this proposal are 1) to examine the cellular and molecular changes in PNNs on PV neurons in the insula after extended binge-like alcohol consumption by mice, and 2) to determine the behavioral consequences of manipulating PV neurons and PNNs on PV neurons in the insula on aversion-resistant drinking. In Specific Aim 1, we will examine the structure of PNNs at different time points after binge drinking using immunocytochemical methods, measure changes in the expression of PNN genes and proteins, and measure the activity of the proteases (MMP and ADAMTS metalloproteinase family) that regulate the remodeling of PNNs. In Specific Aim 2, we will measure changes in synaptic density and volume on PV neurons with and without PNNs after binge drinking using immunocytochemical methods and super-resolution microscopy. In addition, we will measure excitability and synaptic changes on these neurons after binge drinking using electrophysiological methods. Together, these two Specific Aims will provide important knowledge regarding the changes that occur in PNNs and their associated synapses after extended periods of binge alcohol drinking. In the third Specific Aim of this proposal, we will disrupt PNNs on PV neurons by knocking down the expression of two specific PNN proteins encoded by the genes Acan and Bcan using viral-delivered short hairpin RNAs. Viruses will be injected directly into the insula of mice expressing Cre recombinase in PV neurons (PV-Cre) for localized cell-type specific gene knockdown. These mice will be tested for consumption of ethanol alone and an aversive solution of ethanol containing quinine to determine the effect of PNN disruption on aversion-resistant drinking. Finally, we will directly manipulate the activity of insular PV neurons by viral delivery of designer receptors exclusively activated by designer drugs (DREADDs) to determine the role of these neurons in aversion-resistant ethanol consumption. These studies will provide information on the cellular and molecular changes that occur on PV neurons in the insula that drive the transition from binge drinking to high risk drinking and may lead to new therapeutic strategies to treat aversion-resistant drinking.